Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resection
Felix Beuschlein1, Jens Weigel2, Wolfgang Saeger3, Matthias Kroiss4, Vanessa Wild5, Fulvia Daffara6, Rosella Libe7, Arianna Ardito6, Abir Al Ghuzlan8, Marcus Quinkler9, Andrea OBwald1, Cristina L. Ronchi2, Ronald de Krijger1º, Richard A. Feelders11, Jens Waldmann12, Holger S. Willenberg13, Timo Deutschbein2, Anthony Stell14, Martin Reincke1, Mauro Papotti15, Eric Baudin8, Frédérique Tissier16, Harm R. Haak17, Paola Loli18, Massimo Terzolo6, Bruno Allolio2, Hans-Helge Müller19, Martin Fassnacht1,2,4,20
1 Medizinische Klinik and Poliklinik IV, Ludwig-Maximilians-Universität München, Germany; 2 Department of Medicine I, Endocrine and Diabetes Unit, University Hospital, University of Würzburg, Germany; 3 Dept. of Pathology, University of Hamburg, Germany; 4 Comprehensive Cancer Center Mainfranken, University of Würzburg, Germany; 5 Institute of Pathology, University of Würzburg, Germany; 6 Medicina Interna 1, AOU San Luigi and University of Turin, Orbassano, Italy; 7 Département d’Endocrinologie, Groupe hospitalier Cochin, Paris, France; 8 Institut Gustave Roussy, Villejuif, France; 9 Clinical Endocrinology, Campus Mitte, University Hospital Charité, Berlin, Germany; 10 Dept. of Pathology, Erasmus Medical Center, Rotterdam, Netherlands; 11 Dept. of Internal Medicine, Erasmus Medical Center, Rotterdam, Netherlands; 12 Dept. of Visceral,- Thoracic and Vascular Surgery, University Hospital Giessen and Marburg, Campus Marburg, Germany; 13 Dept. of Endocrinology and Diabetology, University of Dusseldorf, Germany; 14 Melbourne eResearch Group, University of Melbourne, Australia; 15 Anatomia Patologica, AOU San Luigi and University of Turin, Orbassano, Italy; 16 Department of Pathology, Pitie-Salpetriere Hospital, AP-HP, Pierre and Marie Curie University, Sorbonne Universités; Inserm U1016, Institut Cochin, Cnrs, UMR8104, Université Paris Descartes, Sorbonne Paris Cité, France; 17 Máxima Medisch Centrum, Eindhoven, Netherlands; 18 Ospedale Niguarda Cà Granda, Milano, Italy; 19 Institute for Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-Universität München, Germany; 2º Central laboratory, University Hospital Würzburg, University of Würzburg, Germany
Background: Recurrence of adrenocortical carcinoma (ACC) even after complete (R0) resection occurs frequently.
Objective: The aim of this study was to identify markers with prognostic value for patients in this clinical setting.
Design, Setting, and Participants: From the German ACC registry 319 patients with ENSAT stage I-III were identified. As an independent validation cohort 250 patients from three European coun- tries were included.
Outcome: Measurements and Statistical Analysis: Clinical, histological and immunohistochemical markers were correlated with recurrence-free (RFS) and overall survival (OS).
Results: and Limitation: While univariable analysis within the German cohort suggested several factors with potential prognostic power, upon multivariable adjustment only a few including age, tumor size, venous tumor thrombus (VTT), and the proliferation marker Ki67 retained significance. Among these Ki67 provided the single best prognostic value for RFS (HR for recurrence 1.042 per 1% increase; p<0.0001) and OS (HR for death 1.051; p<0.0001) which was confirmed in the val- idation cohort. Accordingly, clinical outcome differed significantly between patients with
Copyright @ 2015 by the Endocrine Society Received August 11, 2014. Accepted December 30, 2014.
doi: 10.1210/jc.2014-3182
Abbreviations:
THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
JCEM
EARLY RELEASE:
ENDOCRINE
SOCIETY
Ki67<10%, 10-19%, and ≥20% (for the German cohort: median RFS: 53.2 vs. 31.6 vs. 9.4 months; median OS: 180.5 vs. 113.5 vs. 42.0 months). Using the combined cohort prognostic scores including tumor size, VTT, and Ki67 were established. Although these scores discriminated slightly better between subgroups, there was no clinically meaningful advantage in comparison to Ki67 alone.
Conclusion: This largest study on prognostic markers in localized ACC identified Ki67 as the single most important factor predicting recurrence in patients following R0 resection. Thus, evaluation of Ki67 indices should be introduced as standard grading in all pathology reports of ACC patients.
A drenocortical carcinoma (ACC) is a rare but aggres- sive tumor entity with overall poor prognosis (1-4). Response to medical treatment is limited and only recently the first randomized trial in patients with advanced disease established doxorubicin, etoposide, cisplatin plus mito- tane as first-line cytotoxic therapy (5). However, this trial also demonstrated the limitations of systemic treatment with a median overall survival of only 15 months high- lighting the importance of early diagnosis and appropriate initial treatment. Although strategies of surgical resection such as open or laparoscopic approaches are controversial (6-8), surgery is the mainstay of initial ACC therapy and currently provides the only realistic chance for cure of the disease. However, even after complete resection patients with ACC remain at high risk for recurrence.
As a response to this clinical challenge adjuvant treat- ment with mitotane is frequently recommended (2, 9). Al- though mitotane has shown significant efficacy in pre- venting recurrence in this setting (10), it has a wide range of side effects and impacts hormone (11-13) and drug (14) metabolism. An additional adjuvant measure is irradia- tion of the tumor bed, for which some (15, 16) but not all (17) studies have demonstrated efficacy in preventing local recurrence but not to prolong recurrence-free survival (RFS) or overall survival (OS). Thus, all current treatment concepts have the disadvantages of uncertain efficacy and significant toxicity. Therefore, it would be of major im- portance to limit these treatments to patients with high risk of recurrence which is highly variable in ACC patients (1, 18-21).
While histopathological scores are in use to differenti- ate between benign and malignant adrenal neoplasms they have not been investigated for their prognostic value. Re- cently, a number of molecular markers have been identi- fied that were correlated with clinical outcome (22) and even had predictive value for treatment response (23, 24). However, most applied techniques require fresh frozen tumor material and none of these markers has been eval- uated in a large patient cohort. Therefore, we set out to identify prognostic factors from routine diagnostic work-up to provide guidance for adjuvant therapy after radical resection. For this purpose we took advantage of large cohorts of ACC patients with detailed clinical and
histopathological annotations within the European net- work for the study of adrenal tumors (ENSAT).
Patients and Methods
Patient selection
Patients diagnosed with ACC between 1979 and 2011 were identified from the German ACC registry that fulfilled the fol- lowing inclusion criteria: histologically proven ACC, and local- ized disease (ENSAT stage I-III (18)) after R0 resection. Resec- tion status was judged on the basis of surgical and pathology reports. Furthermore, a minimum follow-up of 12 months was mandatory unless death occurred earlier. Two third of tumor samples of the German cohort were reviewed by the national reference pathologist (W.S.). All items required to calculate the scores suggested by Weiss, van Slooten, and Hough as well as immunohistochemical staining for Mib1 (Ki67) were evaluated. Further clinical information included in the analysis is provided in Table 1+2. Mitotane therapy was defined as adjuvant therapy with mitotane within 3 months following surgery; hormone pro- duction was recorded as any biochemically proven adrenocor- tical hormone excess.
As an independent validation cohort patients from three Eu- ropean countries were identified from the ENSAT ACC registry based on the same inclusion criteria as stated above. Within this cohort a subset of predefined clinical parameters such as age, sex, ENSAT stage (tumor size, lymph node status and VTT), endo- crine activity of the tumor and adjuvant mitotane therapy as well as Ki67 were analyzed.
Both registries had been approved by the local ethics com- mittees of all partaking centers and all included patients had provided written informed consent.
Outcome definitions and statistical analysis
The prespecified primary endpoint of the study was RFS, which was defined as the time interval between initial surgery and the date of radiologic evidence of disease relapse, death re- sulting from any cause or the date of last follow-up. As a sec- ondary endpoint OS was calculated from the date of first surgery to death from any cause or the last follow-up visit. RFS and OS rates over time from initial surgery were estimated using the Kaplan-Meier method. The significance of the demographic pa- rameters and clinical characteristics for prognosis of RFS and OS was determined by univariable and multivariable Cox regression models. As the lowest category for Ki67 < 5% and for tumor size < 5cm was set.
As for the diagnostic scores different strategies were applied to take into account limitations of individual residual analyses. Indeed, following an exploratory analysis model construction
| n | German cohort (n = 319) | n | Validation cohort (n = 250) | |
|---|---|---|---|---|
| Age | 319 | 46.3 (0.4-83.6) | 250 | 46.7 (9.1-83.0) |
| Sex | 319 | 207 females, 112 males | 250 | 162 females, 88 males |
| Median tumor size (cm) | 318 | 10.0 (2.3-40.0) | 248 | 11.0 (2.0-30.0) |
| ENSAT stage (18) | 319 | I: 27 (8.5%) | 245 | I: 22 (9.0%) |
| II: 202 (63.3%) | II: 156 (63.7%) | |||
| III: 90 (28.2%) | III: 67 (27.3%) | |||
| Median Ki67 index (%) | 223 | 10 (1-60) | 239 | 10 (0-82) |
| Adjuvant mitotane therapy | 319 | 84 (26.3%) | 219 | 142 (64.8%) |
| Adjuvant radiation | 313 | 30 (9.6%) | 250 | 7 (2.8%) |
| History of recurrence | 319 | 206 (64.6%) | 250 | 135 (78.4%) |
| Median follow-up of alive patients (months) | 205 | 43.7 | 162 | 69.8 |
| Death from any cause | 319 | 114 (35.7%) | 250 | 88 (35.2%) |
| Death from ACC | 319 | 100 (31.3%) | 250 | 65 (26.0%) |
was based on stepwise, forward and backward selection using p-value criteria in the range between 0.05 and 0.25. Tumor size was explored as a continuous factor and according to cut-off values. Potential cut-off values for tumor size were prespecified at 5cm, 8cm, 11cm, 15cm, and 20cm. Furthermore, the number of factors included, the score statistic, the contribution to the change of the hazard in the Cox-model as well as differentiation of survival curve estimation by use of the predictor were con- sidered. Martingale residual analysis was performed indicating well fitting with continuous Ki67 and exploring grouping by equidistant increases of 5% or of 10%. Extensive sensitivity analyses with multivariable models including, excluding and ex- changing potential factors from the first explored models were performed. Comparisons between groups were conducted ap- plying the log rank test and presenting two-sided p-values. Es- timates of median times to event and hazard ratios (HR) are provided with 95% confidence intervals (CI).
Results
Patient cohorts and characteristics
A total of 319 patients from the German registry and additional 250 patients from 7 European centers fulfilled the prespecified inclusion criteria. Patient characteristics at initial diagnosis are summarized in Table 1. While tu- mor stage and most of other clinical items were not dif- ferent between the groups, the percentage of patients on adjuvant mitotane treatment was significantly lower in the German cohort (26.3 vs. 64.8%).
German cohort
Univariable analyses for all presumably relevant demo- graphic, clinical, and histopathological parameters were performed (Table 2). None of the clinical parameters showed significant correlation with both RFS and OS. Tumor stage was of prognostic value but the discrimina- tion was not found to be sufficient for clinical guidance on adjuvant therapeutic strategies (Table 2 and Supplemental Figure 1). Within the large set of histological parameters
only few were associated with poor clinical outcome (Ta- ble 2) and several multi-item scores were not able to pre- dict outcome better than individual parameters. In con- trast, the proliferation marker Ki67 was found to be the single most relevant predictor of disease recurrence and survival with an hazard ratio (HR) of 1.042 per 1% in- crease of Ki67 index for recurrence (P <. 0001) and 1.051 for death (P < . 0001), respectively (Table 2). Along the same line, Ki67 indices of < 10%, 10%-19%, and ≥20% provided highly significant differences for both RFS (P < .0001) and OS (P <. 0001) translating into a median RFS and a median OS of 53.2 [95% CI 37.7; 74.7] and 180.5 [152.9; no upper limit] months for Ki67 < 10%, 31.6 [21.5; 48.0] and 113.5 [64.4; 153.7] months for Ki67 10%-19%, and 9.4 [7.3; 13.1] and 42.0 [33.7; 56.8] months for Ki67 ≥ 20%, respectively (Figure 1A+C). As inclusion of tumor grading on the basis of mitotic counts had recently been proposed to improve the prediction of prognosis (25) mitotic index was further evaluated. As Ki67 and mitotic index cover similar endpoints in tumor pathophysiology mitotic count expectedly also provided some diagnostic value (Table 2).
Upon a stepwise and a backward multivariable analysis involving all parameters with a p-value of < 0.15 in the univariable analysis plus adjuvant mitotane therapy and taking into account sensitivity analyses and regression di- agnostics, Ki67 remained the factor with the best prog- nostic power with a HR of 1.046 per 1% for RFS and 1.061 per 1% for OS (Table 4). When Ki67 was used as a categorical variable the corresponding HR for RFS was 1.94 [1.25-3.03; P = . 0034] for Ki67 ≥ 10% and 2.58 [1.71-3.92; P < . 0001] for Ki67 ≥ 20% and for OS 3.69 [1.75-7.77; P = . 0006] for Ki67 ≥ 10% and 3.59 [1.99- 6.48; P < . 0001] for Ki67 ≥ 20%. Notably, no other histological parameter retained its relevance when the analysis was adjusted for Ki67. While age, tumor size, and VTT had some association with clinical outcome none of
| Factor [unit] | N of 319 | N+ | RFS | OS | ||||
|---|---|---|---|---|---|---|---|---|
| events | hazard ratio | p-value | events | hazard ratio | p-value | |||
| Age [years] | 319 | 218 | 1.010 per year | 0.0132 | 114 | 1.008 per year | 0.1330 | |
| Tumor size [cm] | 318 | 218 | 114 | |||||
| ≥5 | 293 | 1.276 | 0.3366 | 1.550 | 0.2332 | |||
| ≥8 | 234 | 1.590 | 0.0047 | 1.270 | 0.2853 | |||
| ≥11 | 151 | 1.377 | 0.0189 | 1.153 | 0.4493 | |||
| ≥15 | 68 | 1.477 | 0.0150 | 1.491 | 0.0652 | |||
| ≥20 | 18 | 0.819 | 0.5217 | 0.595 | 0.3087 | |||
| Tumor size 8-20 cm | 318 | 216 | 218 | 1.589 | 0.0025 | 114 | 1.381 | 0.1296 |
| Tumor size 11-20 cm | 318 | 133 | 218 | 1.450 | 0.0066 | 114 | 1.268 | 0.2081 |
| Tumor size 15-20 cm | 318 | 50 | 218 | 1.777 | 0.0009 | 114 | 1.900 | 0.0048 |
| Infiltration in surrounding tissue | 266 | 58 | 179 | 1.236 | 0.2239 | 101 | 1.468 | 0.0823 |
| Invasion in adjacent organ | 271 | 11 | 183 | 1.601 | 0.0080 | 105 | 1.289 | 0.5838 |
| Lymph node positivity | 250 | 20 | 171 | 2.173 | 0.0020 | 97 | 1.846 | 0.0569 |
| Presence of venous tumor thrombus | 306 | 25 | 210 | 1.441 | 0.1215 | 112 | 1.742 | 0.0409 |
| in renal vein or vena cava | ||||||||
| ENSAT stage | 319 | 218 | 114 | |||||
| II or III | 292 | 1.403 | 0.1927 | 1.577 | 0.2150 | |||
| III | 90 | 1.456 | 0.0093 | 1.730 | 0.0042 | |||
| Glucocorticoid secretion | 184 | 101 | 125 | 1.115 | 0.5512 | 65 | 0.715 | 0.1889 |
| Adjuvant mitotane | 319 | 84 | 218 | 0.855 | 0.3402 | 114 | 0.650 | 0.1038 |
| Ki67 [%] | 223 | 143 | 69 | |||||
| ≥5 | 184 | 2.616 | 0.0002 | 4.417 | 0.0015 | |||
| ≥10 | 139 | 2.743 | <0.0001 | 5.322 | <0.0001 | |||
| ≥15 | 82 | 2.810 | <0.0001 | 4.955 | <0.0001 | |||
| ≥20 | 69 | 3.526 | <0.0001 | 5.595 | <0.0001 | |||
| ≥25 | 37 | 3.050 | <0.0001 | 4.320 | <0.0001 | |||
| Ki67 [%] | 223 | 143 | 1.042 per 1% | <0.0001 | 69 | 1.051 per 1% | <0.0001 | |
| Weiss Score ≥5# | 199 | 143 | 138 | 1.435 | 0.0638 | 70 | 1.155 | 0.5810 |
| Mitotic count >5/50HPF | 220 | 154 | 149 | 1.647 | 0.0088 | 77 | 1.357 | 0.2410 |
| Nuclear atypia | 238 | 193 | 162 | 0.675 | 0.0461 | 86 | 0.703 | 0.1877 |
| Atypical mitoses | 227 | 60 | 154 | 0.927 | 0.6819 | 80 | 0.897 | 0.6760 |
| Clear cells <25% | 201 | 183 | 139 | 2.825 | 0.0049 | 70 | 1.650 | 0.2822 |
| Diffuse architecture | 209 | 169 | 141 | 1.648 | 0.0297 | 73 | 0.834 | 0.5157 |
| Venous invasion | 213 | 91 | 147 | 1.758 | 0.0007 | 75 | 1.669 | 0.0286 |
| Sinusoidal invasion | 210 | 123 | 146 | 1.271 | 0.1594 | 74 | 0.988 | 0.9574 |
| Capsular invasion | 271 | 142 | 182 | 1.343 | 0.0489 | 99 | 1.252 | 0.2694 |
| Necrosis | 234 | 189 | 159 | 1.633 | 0.0226 | 83 | 1.830 | 0.0626 |
| Hough score≥3.23# | 189 | 96 | 134 | 1.390 | 0.0001 | 68 | 1.065 | 0.5869 |
| Vascular invasion | 273 | 176 | 187 | 1.462 | 0.0152 | 98 | 1.297 | 0.2289 |
| Fibrous bands | 204 | 121 | 140 | 1.270 | 0.1696 | 69 | 1.275 | 0.372 |
| van Slooten score# | 189 | 96 | 134 | 1.027 | 0.0576 | 68 | 1.002 | 0.9181 |
| Mitotic count >2/10HPF | 220 | 110 | 149 | 1.828 | 0.0003 | 77 | 1.383 | 0.1603 |
| Nuclear hyperchromasia | 231 | 157 | 159 | 0.876 | 0.4347 | 83 | 0.820 | 0.3891 |
| Abnormal nucleoli | 223 | 92 | 155 | 0.804 | 0.1803 | 81 | 0.980 | 0.9275 |
# the cutoff for these scores were set as the median.
N+, number of patients that fulfilled the given criterion.
these factors showed a similar prognostic power for both RFS and OS as Ki67. Similarly, when ENSAT stage was tested within a multivariable model (Table 4) only limited association with RFS or OS was detectable. Of note, ad- juvant treatment with mitotane was not significantly as- sociated with clinical outcome in univariable analysis, but after multivariable adjustment a trend for longer RFS was detectable (HR 0.71; P = . 087) and it became a significant factor for OS (HR 0.41; P = . 009, Table 4).
European validation cohort
Following these observations we investigated a defined subset of clinical and histopathological parameters within the European validation cohort. Comparable to the Ger- man cohort the proliferation marker Ki67 provided a ro- bust prognostic value for prediction of RFS and OS, re-
spectively (Table 3 and Figure 1B+D). Following multivariable analysis Ki67 retained highly significant as- sociation with RFS and OS with a HR for recurrence of 1.020 [1.010-1.029] and for overall survival of 1.026 [1.013-1.039] per 1% increase, respectively (Table 4). Similar to the German cohort, ENSAT stage, age tumor size and the presence of VTT harbored none or only minor prognostic value (Table 4).
Establishment of a prognostic score
In a next step, we aimed at the development of a prog- nostic score for further clinical guidance in the manage- ment of patients with ACC after complete resection. For this purpose, we applied two different models using the pooled data of both cohorts (n = 569). Both algorithms
included the following 3 risk factors (RF): Ki67, tumor size between 15 and 20 cm and presence of VTT.
In the first, basic model Ki67 was regarded as one RF for Ki67 ≥ 10% and as a second RF for Ki67 ≥ 20%. Either tumor size of 15-20cm or presence of VTT was accounted as one combined RF. Each of these 3 RF was counted as 1 point in the prognostic score, which resulted in 4 groups (0-3 RF) with different outcome. However, applying this score, estimated RFS and OS of the different risk groups by Kaplan-Meier analysis provided no clini- cally meaningful separation between the two groups with the highest scores which were therefore combined (Figure 2B, Supplemental Figure 2B). In the next model the same RF were weighted individually according to their prog- nostic power and specifically for RFS. This second mod- eling resulted in a slightly improved risk prediction and allowed for differentiation in more subgroups (Figure 2C, Supplemental Figure 2C).
Discussion
This largest study on prognostic factors in localized ACC provides strong evidence that Ki67 index is the most pow- erful tool of all parameters analyzed in this study to predict recurrence in two independent cohorts of patients after complete surgical resection. Likewise, OS was also strongly associated with the Ki67 index. Following mul- tivariable analysis including age, tumor stage, adjuvant mitotane treatment and all standard histological param- eter used in ACC, the Ki67 index retained its outstanding prognostic power. Importantly, these results initially ob- tained from a German cohort could be validated in an independent European sample. In a next step, we aimed at the establishment of clinical risk scores. Although both applied models including the parameters Ki67, tumor size, and presence of VTT were able to discriminate patient cohorts with different clinical outcome, the added value of these scores in comparison to the use of Ki67 alone was modest. Thus, Ki67 is obviously the best factor to establish a grading system in ACC with Ki67 < 10% defining grade
German cohort
Validation cohort
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| Factor [unit] | RFS | OS | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| N of 250 | N + | events | hazard ratio | p-value | N of 250 | N+ | events | hazard ratio | p-value | |
| Age [years] | 247 | 145 | 1.007 per year | 0.2917 | 249 | 87 | 0.996 per year | 0.6151 | ||
| Tumor size [cm] | 245 | 144 | 247 | 86 | ||||||
| ≥5 | 230 | 1.344 | 0.4803 | 232 | 0.576 | 0.2348 | ||||
| ≥8 | 195 | 2.034 | 0.0051 | 197 | 1.305 | 0.3790 | ||||
| ≥11 | 131 | 1.772 | 0.0012 | 133 | 1.408 | 0.1287 | ||||
| ≥15 | 64 | 1.245 | 0.2268 | 66 | 1.344 | 0.1907 | ||||
| ≥20 | 21 | 0.984 | 0.9540 | 23 | 0.849 | 0.6426 | ||||
| Tumor size 8-20 cm | 245 | 174 | 144 | 1.658 | 0.0111 | 247 | 174 | 86 | 1.302 | 0.2863 |
| Tumor size 11-20 cm | 245 | 110 | 144 | 1.760 | 0.0008 | 247 | 110 | 86 | 1.483 | 0.0690 |
| Tumor size 15-20 cm | 245 | 43 | 144 | 1.361 | 0.1356 | 247 | 43 | 86 | 1.621 | 0.0512 |
| Infiltration in surrounding tissue | 195 | 41 | 124 | 1.100 | 0.6534 | 195 | 41 | 69 | 0.982 | 0.9500 |
| Lymph node positivity | 164 | 9 | 100 | 1.060 | 0.8923 | 164 | 9 | 54 | 2.027 | 0.1058 |
| Presence of venous tumor thrombus | 195 | 23 | 124 | 2.207 | 0.0012 | 195 | 23 | 69 | 2.024 | 0.0237 |
| in renal vein or vena cava | ||||||||||
| ENSAT stage | 242 | 141 | 244 | 84 | ||||||
| II or III | 220 | 1.668 | 0.1617 | 222 | 0.778 | 0.5275 | ||||
| III | 65 | 2.055 | <0.0001 | 67 | 2.391 | <0.0001 | ||||
| Ki67 [%] | 236 | 137 | 238 | 84 | ||||||
| ≥5 | 151 | 2.715 | <0.0001 | 151 | 1.925 | 0.0070 | ||||
| ≥10 | 126 | 2.734 | <0.0001 | 126 | 2.164 | 0.0009 | ||||
| ≥15 | 88 | 3.015 | <0.0001 | 88 | 2.835 | <0.0001 | ||||
| ≥20 | 74 | 2.751 | <0.0001 | 74 | 2.866 | <0.0001 | ||||
| ≥25 | 47 | 2.667 | <0.0001 | 47 | 2.355 | 0.0005 | ||||
| Ki67 [%] | 236 | 137 | 1.024 per 1% | <0.0001 | 238 | 84 | 1.023 per 1% | <0.0001 | ||
| adjuvant mitotane | 218 | 142 | 133 | 1.095 | 0.6209 | 218 | 142 | 77 | 1.054 | 0.8226 |
N+, number of patients that fulfilled the given criterion.
1 tumors, Ki67 10%-19% grade 2 and Ki67 ≥20% grade 3 tumors.
ENSAT stage III has been defined by the presence of positive lymph nodes, tumor infiltration in surrounding tissue or the presence of VTT, while stage IV is restricted to patients with distant metastasis (18). This system which had been independently validated (26) performs well over the whole spectrum of ACC patients to predict overall prognosis. However, as we demonstrate herein within the preselected patient group with localized disease following complete surgical resection the ENSAT staging system seems to be of limited relevance. The reason for this lack of prognostic power is most likely due to the fact that stage III usually comes with a relatively high risk of incomplete resection. In fact, according to a German (27) and an U.S. series (20) this accounts for about 10% of patients with localized ACC. However, these cases as well as those with
uncertain resection status were excluded from our analy- sis, because the high risk for recurrence in these patients is obvious.
Another initially surprising result is the fact that the outcome of patients with a tumor size > 20 cm was better than of those with smaller tumors. However, as described above, this series is a highly selected cohort and very large tumors that are still resectable and not metastasized at the time of surgery might indeed represent a subgroup of tu- mors with specific biological behavior.
To appreciate the quite variable outcome even in the well defined subgroup of completely resected patients, ad- ditional parameters with prognostic value need to be taken into consideration. Recently, inclusion of tumor grading on the basis of mitotic counts has been proposed to im- prove the prediction of prognosis (25). Accordingly, in our two independent cohorts, quantification of Ki67 as a well
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60.
50
Ki67 <10% (n=194)
50
basic RS 0 (n=109)
50
weighted RS 0 (n=98)
40-
40-
40
weighted RS 1 (n=86)
30-
Ki67 10-19% (n=122)
30
basic RS 1 (n=127)
30-
20.
20-
20-
weighted RS 2 (n=110)
10-
10.
basic RS 2 + 3 (n=162)
10-
0-
Ki67 ≥20% (n=143)
0
0-
weighted RS 3 (n=104)
96
90
70
55
39
32
24
20
14
12
9
3
3
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3
194
166
128
97
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67
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99
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45
37
28
23
17
15
11
4
4
4
4
Subjects
86
47
30
25
16
9
8
3
6
3
2
2
Subjects
122
89
2
0
1
1
1
Subjects
127
100
71
63
39
28
18
5
N
3
10
$
2
5 2
1
at risk
110
62
40
23
11
8
5
4
3
3
2
2
at risk
143
85
32
20
9
4
1
1
1
0
at risk
162
73
39
25
12
6
4
2
2
2
2
1
0
104
49
23
17
10
5
3
2
2
2
2
1
0
T
Y
T
0
12
24
36
48
60
72
84
96
108
120
132
144
156
168
180
0
12
24
36
48
60
72
84
96
108
120
132
144
156
168
180
0
12
24
36
48
60
72
84
96
108
120
132
144
156
168
180
Months since RO resection
Months since RO resection
Months since RO resection
defined marker of cellular proliferation provided addi- tional prognostic information with relevant clinical im- pact. This is in good agreement with earlier studies which had suggested Ki67 as a marker with prognostic value in ACC patients (28-31). While these series were small and the overall results therefore not consistent, based on the current results, we strongly suggest tumor grading based on three categories of a Ki67 index < 10%, 10%-19% and above 20%.
As a prognostic factor, Ki67 index in our analysis proved to be superior to different histological scores such as those proposed by Weiss, van Slooten or Hough that are currently in clinical use to differentiate between benign and malignant adrenal tumors. The reason for this finding probably relates to the fact that a number of subitems required for these scores such as atypical mitoses, abnor- mal nucleoli and nuclear atypia had no prognostic poten- tial or were even associated with a trend towards better outcome. This does not question the overall applicability of the scores to discern between benign adrenal adenomas and ACC for which purpose they had been originally pro- posed. However, the findings highlight the limitation of the scores as prognostic tools for this particular group of patients and fuels speculation whether these subitems are of particular importance for the scores. Along the same line, some pathologists have argued for a simplified Weiss score that bases on the more reliable criteria only (32, 33).
The effect of mitotane on clinical outcome was surpris- ingly different between the German cohort and the vali- dation cohort. Due to the retrospective nature of our study we can only speculate about the underlying reasons for this difference. However, the fact that in the German series only 84 out of 319 patients (26%) were treated with mi- totane in comparison to 65% in the validation series al- ready point towards a general difference in therapeutic policies. One explanation for this discrepancy is probably related to the time interval of patient inclusion. Two third of the German patients were diagnosed before 2007, when the land-mark study on the adjuvant usage of mitotane had been published (10), whereas the non-German co- horts were recruited mostly after that time period. How- ever, this observation could also be interpreted as another hint that not all patients will benefit from mitotane treat- ment and prospective trials are required to provide reliable answers.
In variance to recent studies in the current cohort no relevant adverse effect of steroid excess on RFS and OS was found. One of the differences that are present in the current publications refers to the cohorts. While in a recent manuscript by Berruti and colleagues (34) some overlap with our study is present adding patients from North America might have contributed to the observed differ-
ences. In fact, in two studies from single US centers (35, 36) both find Cushing’s syndrome as a marker of poor prog- nosis (Suppl. Table 1). In addition - and probably more importantly - while in our study hormone excess was de- fined by biochemical means, in the published studies this was judged on a clinical basis.
Our study has obvious limitations as a result of its non- randomized design, in which multiple factors may have led to different treatment decisions in individual patients. These limitations are shared by other recent studies that have investigated prognostic factors in ACC patients (for details see Suppl. Table 1). Furthermore, variability of Ki67 index evaluation at different clinical centers is to be expected. This refers to preanalytic variations, usage of different antibodies and staining reagents as well as quan- tification underestimating tumor heterogeneity.
Despite these limitations the high number of patients as well as the inclusion of an independent validation cohort underscores the overall robustness of the reported find- ings. The results could have immediate impact on the clin- ical decision for or against adjuvant treatment options: even after complete resection patients with high Ki67 in- dex have a high likelihood to suffer from recurrent disease, thereby calling for a more aggressive therapeutic course. In contrast, patients with a low Ki67 index are likely to have a less favorable risk/benefit ratio of adjuvant treatment considering its substantial toxicity. Whether or not mito- tane is the appropriate treatment particularly for tumors with low proliferation rate remains open. At least part of this question will be answered by the ongoing ADIUVO trial (mitotane vs. observation in low grade tumors after R0 resection; www.adiuvo-trial.org)
Conclusions
In conclusion, in this study analyzing multiple potential prognostic markers in two independent cohorts of 568 patients with completely resected ACC, Ki67 emerged as the single most important factor predicting recurrence and should be part of any pathology report of ACC to provide its proliferative potential. This finding will further guide the management of patients with this rare disease.
Acknowledgments
This project has been supported by the European Network for the Study of Adrenal Tumors (ENS@T). The research leading to these results has received funding from the Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 259 735.
The authors are indebted to Stefanie Hahner, Michaela Haaf,
| Factor [unit] | N+ | Recurrence-free survival | Validation cohort | |||||
|---|---|---|---|---|---|---|---|---|
| German cohort | ||||||||
| hazard ratio | 95% CI | p-value | N+ | hazard ratio | 95% CI | p-value | ||
| ENSAT stage | ||||||||
| II | 202 | 1.257 | 0.747-2.115 | 0.3885 | 155 | 1.342 | 0.647-2.783 | 0.4291 |
| III | 90 | 1.780 | 1.034-3.063 | 0.0373 | 65 | 2.675 | 1.266-5.652 | 0.0099 |
| Age [years] | 1.013 | 1.004-1.022 | 0.0054 | 1.001 | 0.986-1.016 | 0.8961 | ||
| Tumor size 15 - 20 cm | 34 | 1.601 | 1.033-2.480 | 0.0354 | 38 | 1.369 | 0.876-2.138 | 0.1678 |
| Presence of venous tumor thrombus | 15 | 1.327 | 0.724-2.432 | 0.3599 | 23 | 1.828 | 1.111-3.008 | 0.0176 |
| in renal vein or vena cava | ||||||||
| Ki67 [%] | 1.046 | 1.033-1.059 | <0.0001 | 1.020 | 1.010-1.029 | <0.0001 | ||
| Adjuvant mitotane | 63 | 0.705 | 0.473-1.052 | 0.0867 | 117 | 0.966 | 0.654-1.426 | 0.8617 |
| Overall survival German cohort | Validation cohort | |||||||
| Factor [unit] | N+ | hazard ratio | 95% CI | p-value | N+ | hazard ratio | 95% CI | p-value |
| ENSAT stage | ||||||||
| II | 202 | 1.290 | 0.615-2.705 | 0.5002 | 155 | 0.540 | 0.240-1.213 | 0.1356 |
| III | 90 | 2.155 | 1.018-4.562 | 0.0449 | 67 | 1.389 | 0.618-3.122 | 0.4268 |
| Age [years] | 1.014 | 1.001-1.028 | 0.0314 | 0.990 | 0.970-1.010 | 0.3325 | ||
| Tumor size 15 - 20 cm | 34 | 1.192 | 0.632-2.251 | 0.5872 | 38 | 1.830 | 1.070-3.128 | 0.0273 |
| Presence of venous tumor thrombus | 15 | 2.141 | 1.075-4.265 | 0.0303 | 23 | 1.438 | 0.762-2.712 | 0.2622 |
| in renal vein or vena cava | ||||||||
| Ki67 [%] | 1.061 | 1.044-1.079 | <0.0001 | 1.026 | 1.013-1.039 | <0.0001 | ||
| Adjuvant mitotane | 63 | 0.410 | 0.211-0.797 | 0.0086 | 117 | 0.804 | 0.482-1.343 | 0.4053 |
* the analyses include only patients for whom all parameters were available (ENSAT: n = 319 for the German cohort, of whom 218 experienced recurrence and 114 died and with 141 recurrences of n = 242 and 84 deaths of n = 244 for the validation cohort; other most relevant factors: n = 214 for the German cohort, of whom 139 experienced recurrence and 68 died and n = 181 with 114 recurrences and 65 deaths for the validation cohort)
N+, number of patients that fulfilled the given criterion.
and Margarita Bala (Department of Medicine I, Endocrine and Diabetes Unit, University Hospital, University of Würzburg, Germany) and Nicole Reisch, Brigitte Mauracher and Susanne Schmid (Medizinische Klinik and Poliklinik IV, Ludwig-Maxi- milians-Universität München, Germany) for help in patient re- cruitment, data management, and valuable input to the manu- script, respectively.
Address all correspondence and requests for reprints to: Cor- responding author: Martin Fassnacht, MD, Schwerpunkt En- dokrinologie, Medizinische Klinik und Poliklinik I des Univer- sitätsklinikums, Zentrum Innere Medizin, Oberdürrbacher Straße 6, D-97 080 Würzburg, Germany, Fon: +49-931-201- 39 021, Fax: +49-931-201-60 39 021, Mail: Fassnacht M@ukw.de.
Disclosure Summary: The authors have nothing to disclose This work was supported by .
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